Methods have been conventionally proposed for observing the shape of an object disposed on the surface of a substrate by irradiating the object with an excitation light. For example, in a device disclosed in Patent Document 1, an evanescent wave is generated on the surface of a transparent substrate by irradiating the substrate with an excitation light output from an excitation light source and causing the total reflection of the excitation light therein. Then, a scattered light of the evanescent wave generated by a specimen on the substrate is detected. The device disclosed in Patent Document 1 does not disperse the scattered light.
For example, Patent Document 2 discloses a device for dispersing a fluorescence and a scattered light emitted from a specimen component excited by an evanescent wave. In the device disclosed in Patent Document 2, the specimen component is not immobilized on an interface of a flow path.
On the other hand, a device is proposed which is designed to immobilize a plurality of biomolecules on the surface of a substrate, and to generate an evanescent wave in a certain range of the substrate surface like Patent Document 1 to thereby make an image of light emission from the biomolecules excited by the evanescent wave. Non-fluorescent biomolecules are immobilized on the substrate, and a reaction solution containing fluorescent molecules is made to flow onto the substrate, whereby the fluorescence generated from the position where the biomolecules are immobilized is observed. This can observe a bonding reaction between the biomolecules and the molecules in the reaction solution. For example, first, an unlabeled single-stranded DNA is immobilized on the substrate, into which a reaction solution is introduced. The reaction solution contains fluorescence labeled bases which are labeled by different fluorescent substances according to the type of the base. The fluorescence generated from the position of immobilizing the molecule is dispersed, while bonding a complementary base to the single-stranded DNA, so that the sequence of the immobilized DNA can be determined.
Recently, as disclosed in Non-Patent Document 2, methods are proposed for determining the sequence of bases of the DNA by immobilizing the DNA on a substrate. The molecules of specimen DNA fragments to be analyzed are captured on the substrate one by one in a random manner, and each molecule is extended substantially by one base. The result is detected by measuring the fluorescence to thereby determine the sequence of bases. Specifically, four types of dNTP derivatives (MdNTP) with detectable labels are captured into a template DNA as a substrate of a DNA polymerase, and then can terminate a DNA chain elongation reaction under the presence of a protective group. In a step of a DNA polymerase reaction, the DNA polymerase reaction is performed by using these four types of dNTP derivatives. Then, in a step of detection, the captured MdNTP is detected by fluorescence or the like. In a step of returning, the MdNTP is returned to a state of being extendable. These steps are set as one cycle and repeatedly performed, so that the sequence of bases of the specimen DNA is determined. Since in this technique the base sequence of the DNA fragments can be determined by each molecule, a number of fragments can be simultaneously analyzed, which can increase the throughput for analysis. Since in this system the base sequence can be determined for each single DNA molecule, cloning which was a problem of the related art, and the purification and amplification of the specimen DNA at a PCR or the like possibly become unnecessary, so that the acceleration of genomic analysis or genetic diagnosis can be expected. This method immobilizes the specimen DNA fragment molecules to be analyzed on the substrate surface in a random manner, and thus needs an expensive camera with the number of pixels which is several hundreds times larger than the number of the captured DNA fragment molecules. That is, when a distance between the DNA fragment molecules is adjusted to an average of 1 micron, some DNA fragment molecules have a distance therebetween more than the average, and other DNA fragment molecules have a distance therebetween less than the average. In order to detect the DNA molecules by separation of them, it is necessary to detect fluorescent images at narrower intervals in terms of a substrate surface. Normally, the images are required to be measured at intervals of one several tenth of the distance.
On the other hand, Non-Patent Document 3 and Patent Document 3 employ a nano-opening evanescent illumination detecting system which can further reduce a volume of irradiation of an excitation light rather than in a total reflection evanescent illumination detecting system to thereby improve the sensitivity of detection of the fluorescence. Two glass substrates, namely, a glass substrate A and a glass substrate B are disposed in parallel. A plate-like aluminum thin film of about 100 nm in thickness with a nano-sized opening of 50 nm in diameter is deposited on one side of the glass substrate opposed to the glass substrate B. The aluminum thin film needs to have an optical-shielding characteristic. A reaction chamber is provided in an intermediate position between the two glass substrates, and a solution is charged into the reaction chamber, whereby a solution layer is formed between the two glass substrates. The reaction chamber has an inlet and an outlet for the solution. The solution is charged from the inlet and discharged from the outlet, so that the solution can flow in parallel to the glass substrates and the aluminum thin film. Thus, the composition of the solution of the solution layer can be changed to an arbitrary one. When the excitation light having a wavelength of 488 nm and generated from an Ar ion laser is applied vertically to the glass substrate A by stopping down the aperture of an objective lens in the direction opposite to the glass substrate B, an evanescent field of the excitation light is formed in the solution layer near the bottom plane of the inside of the nano-sized opening. Thus, the excitation light is not transferred anymore to the further inside of the solution layer. In contrast, the emission of fluorescence is detected by forming an image on a two-dimensional CCD using the objective lens. The intensity of excitation light in the evanescent field is attenuated in an exponential manner with increasing distance from the bottom plane of the nano-sized opening. The intensity of excitation light becomes one tenth ( 1/10) at a distance of about 30 nm from the bottom plane of the nano-sized opening. Further, in the nano-opening evanescent illumination detecting system, unlike the total reflection evanescent illumination detecting method, the width of irradiation of the excitation light in the parallel to the glass substrate is limited to the diameter of the opening, namely, 50 nm, which further reduces the volume of irradiation of the excitation light. Thus, the fluorescent emitted from a free fluorescent substance and a background light including Raman scattering of water can be drastically reduced. As a result, under the presence of the free fluorescent substances at a higher concentration, only the fluorescent substances marked in biomolecules of interest can be selectively detected, which can achieve the detection of fluorescence with very high sensitivity.
In the invention, the above fluorescence detecting system can be applied to measurement of the captured dNTP in the elongation reaction of the DNA molecule. In the same manner as the term “specimen component immobilized surface”, the plane where the evanescent field is generated can be referred to as an “evanescent field interface surface”.